The primary focus of our research is the characterization of methylenetetrahydrofolate reductase (MTHFR). This protein is an enzyme that is involved in the generation of the methyl group of methionine in E. coli, humans, and other organisms. MTHFR deficiency in humans can result in homocysteinemia, an autosomal recessive disorder. Homocysteinemia is characterized by varying phenotypes such as developmental delay, motor and gait abnormalities, seizures and psychiatric disturbances. In addition, homocysteinemia has recently been shown to be a significant risk factor for premature cardiovascular disease. In E. coli, the metF gene codes for the MTHFR protein. The E. coli MetF shows significant homology to the N-terminal region of the human MTHFR. Mutations recently identified in humans with MTHFR deficiency occur in regions of high homology with MTHFRs from other species, and these mutations are associated with loss of enzymatic activity. We have, therefore, decided to use the human mutations as a guide to the mutational analysis of the E. coli MetF. Because these mutations occur in evolutionarily conserved regions, insights into requirements for flavin and folate binding, and catalysis of reduction can be obtained by biochemical characterization of the mutant proteins.